There is growing evidence that the immune system is actively involved in metabolic regulation under both normal and pathophysiological conditions. Our laboratory recently identified a novel splice variant of the -subunit of thyroid stimulating hormone (TSH) - the first functional TSH isoform to be identified. Preliminary and published findings demonstrate that this splice variant is preferentially produced by cells of the immune system; that bone marrow (BM)-derived leukocytes traffic to the thyroid where they produce elevated levels of the splice variant in response to systemic virus infection; and that in vivo exposure of mice to recombinant splice variant protein curtails circulating thyroid hormone levels. Additionally, an shRNA that targets the TSH splice variant blocks expression of the splice variant but not the native form of TSH. The goal of this R03 grant is to make mice in which the TSH splice variant is suppressed in cells of the immune system. Preliminary studies using those animals will form the basis of a future R01 research proposal. There are two specific aims: Aim 1: To make a lentivirus/TSH- shRNA vector (LV-TSH-shRNA) that targets and suppresses the TSH isoform. LV-TSH- shRNA vectors will be made using protocols that are familiar to our laboratory. These will be tested in vitro for their ability suppress the TSH splice variant using established cell lines and in bone marrow hematopoietic cells. Aim 2 To make radiation chimeras using BM cells transduced with the LV-TSH-shRNA vector in order to suppress the splice variant in cells of the immune system. BM cells will be transduced with the LV-TSH-shRNA vector and enriched by cell sorting based on GFP expression (for identification of transduced cells) and CD34 expression (as a marker of early BM stem cells). These cells will be used to immunologically reconstitute irradiated syngeneic mice. Mice will be studied for their ability to respond to bacterial and viral infection. Findings from these studies will form the basi for a future R01 application aimed at understanding how the immune system contributes to the control of the metabolic response during infection and inflammation. PUBLIC HEALTH RELEVANCE: Although it has been known for many years that the immune system and the endocrine system interact collaboratively, the functional significance of this has remained obscure. In these studies, we will explore the mechanisms by the immune system regulates thyroid hormone levels using a recently identified new form of thyroid stimulating hormone.